No expression of his-tagged protein - (Dec/20/2005 )

Hello,

One of the proteins I am working on doesn't express at all. I have tried multiple cell strains such as BL21 De3, rosetta, origami, rosetta gami, rosetta pLysS etc but I never acheive expression. The protein is 21kDa and His tagged. The plasmid was sequenced and showed no mutations or problems. I have also tried expressing at different temperatures, for different amounts of time, and using IPTG and even autoinduction. It is possible that this proten is toxic in e.coli, however the cells appear to grow normally and the pLysS strains make no difference. What could be happening?

Thanks.

-aer500-

QUOTE (aer500 @ Dec 20 2005, 04:29 PM)

Hello,

One of the proteins I am working on doesn't express at all. I have tried multiple cell strains such as BL21 De3, rosetta, origami, rosetta gami, rosetta pLysS etc but I never acheive expression. The protein is 21kDa and His tagged. The plasmid was sequenced and showed no mutations or problems. I have also tried expressing at different temperatures, for different amounts of time, and using IPTG and even autoinduction. It is possible that this proten is toxic in e.coli, however the cells appear to grow normally and the pLysS strains make no difference. What could be happening?

Thanks.

Well as I already answered many times on the forum "every protein is another world" that means that you can use exactly the same protocol and never get expression

I just gave up on two different Myccobacteria protein cause I couldn't get them expressed even with the same type of trials than you. I guess ut's just that way some works other not ! It can be due to many reason, association with the cell wall, strange codons usage, toxicity, pseudo gene etc...

Do you have any informations on your protein ? is it very Hydrophobic in terms of amino acids residue ? usually it's not a very good pronostic for succesfull expression !

Did you check the neighbouring amino acids around the Met ? Some are really known to induce instability

You could maybe try to do a truncated version of your protein including your important motif !

Pesji

-pesji-

To this day I still dont fully understand why I could get my 7kDa and 14kDa his-tagged proteins to express.

Fo weeks I'd tried absolutely everything from temp, timepoint and altering IPTG then one day the incubator I'd been using was busy so I had to use another one. This incubator had no rpm speed dial so I just did what I thought, (which now I look at and realise its heaps faster than the 200-225rpm I was using on the other and quite rough in the way it shakes)..... and I got heaps of expression (yet my control for protein expression no longer worked.... so I agree with the above response)

At the time I thought it must have been a temp difference between the two incubators but it wasnt. Then I spoke to some other people about it and they agreed that shaking the crap out of the culture for some reason can improve expression. Something to do with aeration. Also decreasing the volume of media per culture flask can help (also to do with aeration). Mine works with 35ml culture per 250ml culture flask...so I just grew up 225 ml and at the same time as IPTG induction I split into 35ml cultures and it works just fine.

Anyways it just a consideration that I had overlooked completely.

-dhc200040-

QUOTE (aer500 @ Dec 20 2005, 04:29 PM)

Hello,

One of the proteins I am working on doesn't express at all. I have tried multiple cell strains such as BL21 De3, rosetta, origami, rosetta gami, rosetta pLysS etc but I never acheive expression. The protein is 21kDa and His tagged. The plasmid was sequenced and showed no mutations or problems. I have also tried expressing at different temperatures, for different amounts of time, and using IPTG and even autoinduction. It is possible that this proten is toxic in e.coli, however the cells appear to grow normally and the pLysS strains make no difference. What could be happening?

Thanks.

hi aer500,

i have had the same problems overexpressing a protein. also testing different strains, incubation times and induction conditions - but no expression occured. plasmid was also sequenced but the sequence was still in frame with no mutations or other things... but then i have changed the expression vector-system, subcloned my insert into another plasmid and now i get a great overexpression of my protein....c'est la vie... hope it helps a little

-flausch-

Thank you all for your help. I think I will try purifying some plasmid from the expression cells and send it for sequencing and see if anything strange has happened.